Chronic rejection is the principal obstacle to long-term survival and function of heart allografts, both clinically and in our cynomolgus monkey non-human primate model. This application will test the hypothesis that pathogenic mechanisms driven by the CD28/B7, CD40/CD154, and ICOS costimulation pathways mediate CAV, and constitute the principal barrier to tolerance induction for a heart allograft. This hypothesis is strongly supported by our findings during the first term of this award mechanism. When combined with either a calcineurin inhibitor (CNI: cyclosporine A) or CD154 blockade, selective targeting of CD28 for three weeks after transplant significantly inhibited alloantibody elaboration and CAV at 90 days after transplant. Similarly, pre-emptive B-cell depletion with ?CD154 or CNI significantly inhibited pathogenic alloimmunity. This application will extend these observations and evaluate whether the ?CD28/B7- and ?CD40/CD40L-based costimulation pathway blocking regimens that appear most promising in our work to date will consistently induce tolerance to a cardiac allograft one year after cessation of treatment. Specifically, the durability and failure mode of our most promising regimens (anti-CD28 with additional CD40/CD154 blockade; ?CD28 induction with delayed CNI withdrawal) will be established. In addition, selective ?CD28 with preemptive B-cell depletion and CD40 blockade or depletion to replace ?CD154 will be systematically evaluated for their tolerogenic potential. We predict that more effective control of pathogenic costimulation pathway activation will prevent alloantibody elaboration and CAV progression after treatment ends, and prevent loss of unresponsiveness to cardiac myosin, a heart-specific autoantigen. If so, we predict that graft acceptance and operational tolerance will also be observed in otherwise immunocompetent monkeys, as gauged by their responses to a panel of vaccine antigens. By measuring how alloimmunity and loss of tolerance to autoantigens are modulated by these candidate tolerance-inducing regimens, we will identify pathways resistant to costimulation-based tolerance induction and assess the biological and molecular correlates of graft injury or acceptance.